Creatine kinase (CK) is an essential enzyme involved in the regulation of energy metabolism in muscle, brain, and other tissues. Because of the presence of reactive thiol groups in its active center, CK is vulnerable to inactivation by oxidants. Peroxynitrite is a product of the reaction between superoxide and nitric oxide which is formed in vivo in many cell types. We have used CK from rabbit muscle, and 3-morpholinosydnonimine (SIN-1) which decomposes to release superoxide and nitric oxide forming peroxynitrite. SIN-1 (100 micromole) inhibited CK to 1.9% of control within 20 min, and thiol reducing agents glutathione (1 mM) and dithiothreitol (1 mM) repaired the activity of inhibited enzymes to 22+/- 4 and 17+/- 2% of control, respectively. However, the activity of CK inhibited with S-nitrosothiols and hydrogen peroxide was recovered completely by thiol reducing agents. Incubation of CK with SIN-1 and superoxide dismutase (500 U/ml) prevented the SIN-1 induced inhibition indicating that nitric oxide itself did not inactivate the enzyme. SIN-1 induced inhibition of CK was also prevented by nitric oxide donor, spermineNONO (3-25 micromole), but spermine hydrochloride was not effective. Superoxide radical generating system (xanthine+xanthine oxidase) moderately inhibited CK, and addition of nitric oxide to this incubation mixture to form peroxynitrite exacerbated the CK inhibition. We conclude that peroxynitrite is a mainly irreversible inhibitor of CK, and is more potent than either superoxide or hydrogen peroxide.